Pharmaceutical compositions containing IL-6

ABSTRACT

Pharmaceutical compositions based on Interleukin-6 (IL-6) stabilized with non reducing sugars, such as sucrose and trehalose. The compositions may also contain an amino acid or human albumin as an excipient. The formulation is particularly suitable for the stabilization of recombinant IL-6 freeze-dried powder.

The present invention contemplates pharmaceutical compositionscontaining Interleukin-6 (IL-6), and particularly contemplatescompositions based on IL-6 stabilized with nonreducing sugars.Interleukin-6 is a protein belonging to the group of cytokines, whichproved to play a key role in the organism immune response andhaematopoiesis stimulation (International Symposium on IL-6:Physiopathology and Clinical Potentials, Montreux, Oct. 21-23, 1991).

The prospective therapeutic applications of IL-6 are tumoral growthinhibition, treatment of thrombocytopenia caused by chemotherapy,radiotherapy, and even accidental exposure to radiations. It may also beused as a vaccine adjuvant.

According to the present invention, IL-6 may be either natural orsynthetic, i.e. produced on the basis of recombinant DNA technology, thelatter being preferred.

The protein of this invention is glycosylated human IL-6, prepared onthe basis of the recombinant DNA technology by expression in CHO(Chinese Hamster Ovary) cells, transformed with the corresponding DNA,according to the disclosures of European Patent Application EP 0220574.

As known, purified proteins show a great tendency to become denaturated,even by normal atmospheric agents. This characteristic is even moreevident in proteins produced on the basis of recombinant DNA technology.To prevent any contamination of non-human origin, they must be purifiedto a high degree, which makes their stability lower than that ofcorresponding purified natural proteins.

IL-6 formulations for injection are obtained on the basis of a processinclusive of freeze-drying for dry powder production.

As described by M. J. Pikal in Biopharm., Oct. 25-30, 1990, the proteinpharmacological activity is reduced by phenomena taking place duringfreeze-drying.

For example, proteic aggregates, which are generally regarded asdirectly responsible for the onset of allergic manifestations,frequently form during the process. Furthermore, should the protein benot damaged by the various process stresses, a partial denaturation ofsame during storage operations would be extremely probable.

It is just because of the very easy denaturation of highly purifiedproteins that it is highly desirable to produce stable formulations withan as long life cycle as possible, even when stored at ambienttemperature.

The expression "formulation stability" is used to mean that the proteinmaintains its activity both during the pharmaceutical preparation andstorage.

The formulations containing highly purified proteins may be stabilizedby addition of one or more excipients preventing or delaying the activeingredient degradation.

Excipients of different chemical nature were used in various proteinsformulations.

High molecular weight stabilizers of biological origin, such as seacolloids, dextran, and phospholipids, are known.

Equally effective stabilizers often proved to be the formulationscontaining proteins, e.g. albumin, amino acids, e.g. arginine orglycine, and sugars, e.g. monosaccharides or oligosaccharides. Anothercytokine, i.e. Interleukin-2 (IL-2), and particularly its recombinantform, was formulated with various stabilizers, preferably albumin andamino acids.

International patent application WO 90/00397 discloses IL-2stabilization with arginine or carnitine or a mixture thereof, withbetaine, pyridoxine, polyvinylpyrrolidone, carboxylic acids salts, andby the addition, if any, of other excipients, such as sugars and citratebuffer.

European patent application EP 158487 discloses IL-2 formulations withhuman albumin and a reducing compound, such as glutathione,N-acetylcysteine or ascorbic acid.

Pikal in Biopharm., Oct. 25-30, 1990, also suggests that excipientscapable of bringing about amorphous and/or vitreous structures can causeprotein stabilization on drying.

The amorphous structure seems to secure a considerable restriction ofprotein molecular mobility, with consequent decrease in chemicalreactivity, as well as a long lasting protection: in fact, it issupposed to form a sort of casing where the protein is housed and,therefore, protected also after the process cycle.

However, Pikal states that an amorphous excipient is not sufficient forstability increase. Actually, the protein may be denaturated just byinteracting with the amorphous excipient.

The conclusion is that a general criterion for proteins formulationcannot be put forward: the optimal formulation composition can bedetermined only through an exacting work of screening of a large numberof substances.

The study of a new protein, such as IL-6, required an in-depthinvestigation of various stabilizing agents, including the substancesthat give an amorphous structure, such as nonreducing sugars.

It has surprisingly been found that nonreducing sugars, such as forexample sucrose and trehalose, increase the stability of IL-6formulation.

It is the main object of the present invention to provide apharmaceutical composition containing an intimate mixture of IL-6 and astabilizing quantity of a nonreducing sugar either alone or inconjunction with other excipients.

It is a further object of the present invention to provide a procedurefor the preparation of said pharmaceutical composition, including thecomponents for aqueous solution freeze-drying.

It is a further object of the present invention to provide a form ofsaid pharmaceutical composition in which the aforesaid intimate solidmixture is hermetically enclosed in a sterile container suitable forstorage before use and for the mixture reconstitution in a solution forinjection.

It is a further object of the present invention to provide a solution ofsaid solid mixture reconstituted in a solution for injection.

With a view to evaluating the excipient effect on the active ingredientstability, several formulations of recombinant IL-6 containing 35μg/vial were prepared with various excipients, such as mannitol,sucrose, trehalose, lactose mixed with an amino acid, such as arginineor glycine, or with human serum albumin (HSA). Table 1 shows thecomposition of the various formulations prepared (A1, A2, A3, A4, etc.),expressed as content (in mg) per vial. All formulations contain arginineor glycine or human serum albumin (HSA) in addition to other excipients.

                                      TABLE 1                                     __________________________________________________________________________    Formulations of recombinant IL-6 (35 μg)                                   (content/vial)                                                                COMP. HSA Mannitol                                                                            Saccharose                                                                           Trehalose                                                                            Lactose                                                                             Arginine HCl                                                                          Glycine                           FORM. mg  mg    mg     mg     mg    mg      mg    Na.sub.2 HPO.sub.4                                                                   Na.sub.2                                                                      HPO.sub.4            __________________________________________________________________________    A1    0.2 25                        0.125         0.313  0.336                A2    0.2 25                                0.5   0.313  0.336                A3    0.2       47.5                0.125         0.313  0.336                A4        25                        0.125         0.313  0.336                A5              47.5                0.125         0.313  0.336                A6                     47.5         0.125         0.313  0.336                A7        23.7                      1.5           0.313  0.336                A8              44.5                1.5           0.313  0.336                A9                            44.5  1.5           0.313  0.336                A10                                 1.5     10.4  0.313  0.336                __________________________________________________________________________

The freeze-dried powder was obtained on the basis of the followingprocess: IL-6 bulk was diluted with the excipient solution in phosphatebuffer at pH 7. The solution obtained was filtered, made up to volume,poured into the vials, and freeze-dried.

The samples were maintained at 50° C. and subjected to immunologic- andbioassays at set time intervals.

The immunologic assay was carried out using QUANTIKINE kit, (R&D SYSTEMSInc.), cat. No. D6050, following the instructions attached thereto.

The bioassay was carried out as described by Normann and Potter inScience, 233, 566-569, 1980. The assay measures IL-6 activity byexploiting IL-6 capability of acting as a growth factor of a particularcell line (plasmacytoma T-1165).

Activity is expressed in international units/solution milliliter(IU/ml).

An international unit is the quantity of IL-6 producing 50% of maximumcell growth.

In this specification, the measure is expressed as per cent recovery ofthe activity of sample IL-6 in the various formulations, on theassumption that the sample activity at zero time is 100%. Assays werecarried out in duplicate.

Tables 2 and 3 show the results of assays conducted on the samples ofTable 1 after 4, 5, 7, 8 and 9 weeks (Table 2) and after 10, 12, and 21weeks (Table 3).

Samples A1 to A6 were subjected to immunologic assay (Table 2) andsamples A7 to A10 were subjected to bioassay (Table 3).

                  TABLE 2                                                         ______________________________________                                        Stability at 50° C. of IL-6 formulations A1 to A6 (35 μg)           by immunologic assay, expressed as % recovery vs. zero time                            50° C.                                                        Formulation                                                                              4W        5W     7W     8W   9W                                    ______________________________________                                        A1                          62     80   84                                    A2                          78     80   80                                    A3                          103    120  112                                   A4         81        82                                                       A5         107       104                                                      A6         89        100                                                      ______________________________________                                         W = weeks                                                                

                  TABLE 3                                                         ______________________________________                                        Stability at 50° C. of IL-6 formulations A7 to A10 (35 μg)          by bioassay, expressed as % recovery vs. zero time                                      50° C.                                                       Formulation 10W          12W    21W                                           ______________________________________                                        A7          38           35     37                                            A8          104          95     74                                            A9          51           49                                                    A10        56           61                                                   ______________________________________                                         W = weeks                                                                

The data shown in the Tables reported above demonstrate that thecompositions containing nonreducing sugar, such as e.g. sucrose ortrehalose, (A3, A5, A6, A8) are much more stable than the compositionscontaining mannitel or lactose (A1, A2, A4, A6, A7). With a view toevaluating the effect of arginine, glycine or albumin on theformulations stability, IL-6 compositions containing sucrose of lactosealone vs. compositions containing the additional excipient were prepared(Table 4).

For the purpose of evaluating the effect of pH on the stabilizing actionof the various components, the formulations were prepared byfreeze-drying aqueous solutions at various pH (5.5, 6, and 7).

                                      TABLE 4                                     __________________________________________________________________________    Recombinant IL-6 formulations (35 μg) containing sucrose or                lactose with or without additional excipient (content/vial)                   Comp.                                                                             Saccharose                                                                          Lactose                                                                            Arginine HCl                                                                         HSA                                                                              Na.sub.2 HPO.sub.4                                                                  NaH.sub.2 PO.sub.4                             Form.                                                                             mg    mg   mg     mg mg    mg    pH                                       __________________________________________________________________________    B1  45                   0.035 1.17  5.5                                      B2        45             0.035 1.17  5.5                                      B3  40.4       1.5       0.035 1.17  5.5                                      B4        40.4 1.5       0.035 1.17  5.5                                      B5  45                0.25                                                                             0.035 1.17  5.5                                      B6  45                   0.107 1.11  6.0                                      B7        45             0.107 1.11  6.0                                      B8  40         1.5       0.107 1.11  6.0                                      B9        40   1.5       0.107 1.11  6.0                                      B10 45                0.25                                                                             0.107 1.11  6.0                                      B11 48                   0.313 0.336 7.0                                      B12       48             0.313 0.336 7.0                                      B13 43.3       1.5       0.313 0.336 7.0                                      B14       43.3 1.5       0.313 0.336 7.0                                      B15 48                0.25                                                                             0.313 0.336 7.0                                      __________________________________________________________________________

The stability of the above formulations was studied on samplesmaintained at 25° C. and 50° C.; the residual activity was measured atthe time intervals shown in Tables 5 and 6. Table 5 illustrates thestability data of samples subjected to immunologic assay and Table 6shows the stability data of samples subjected to bioassay. Activity dataare expressed as % recovery vs. zero time.

                                      TABLE 5                                     __________________________________________________________________________    Comparison among stability data of IL-6 formulations (35 μg)               containing sucrose or lactose with or without an additional                   excipient. (% recovery vs. zero time) - immunologic assay                     25° C.               50° C.                                     Form.                                                                             2W 3W 4W 6W 7W 8W 9W 10W                                                                              2W 3W 4W 6W 7W 8W 10W                             __________________________________________________________________________    B1  98       112            112      103                                      B2           92             99       89                                       B3  86       96             129                                               B4  90                      91       72                                       B5  91       86             89       83                                       B6        112                     104   101                                   B7        89    74                71                                          B8        115   93                112   107                                   B9        97    90                97    110                                   B10 88       85                95                                             B11       91    116                                                                              98    119      95    120   101                             B12       105            95       103         87                              B13       107      102   100      87       80                                 B14       103      86    92       94    119                                   B15    107      11    103      105                                            __________________________________________________________________________     W = weeks                                                                

                                      TABLE 6                                     __________________________________________________________________________    Comparison among stability data of IL-6 formulations (35 μg)               containing sucrose or lactose with or without an additional                   excipient. (% recovery vs. zero time) - bioassay                              25° C.               50° C.                                     Form.                                                                             2W 3W 4W 5W 6W 7W 8W 9W 2W 3W 4W 5W 6W 7W 8W 9W                           __________________________________________________________________________    B1  107         96    100   93    105         91                              B2  109         94    86    99    85          75                              B3  117         98          105   93    103                                   B4  90                96    104   94          77                              B5  94    103         81    100   98          93                              B6  92       96 108         96 106         95    103                          B7  110         84          104      83    70                                 B8  113      109                                                                              103      120                                                                              118                                                                              119      106      118                          B9  93       72             80 75    68                                       B10 106               109   103   92          112                             B11    106   98          111                                                                              115      113         104                          B12    110   81          102                                                                              81       74          70                           B13    94          97       88             95                                 B14    94          103      95       70    73    69                           B15 97                110         89          102                             __________________________________________________________________________     W = weeks                                                                

As may be seen, the further excipients, i.e. arginine and albumin, addedto the formulations containing sucrose and lactose participate in thestabilizing action to a negligible extent.

The data listed in Tables 5 and 6 also demonstrate that the formulationscontaining a nonreducing sugar, e.g. sucrose, show a much lowerdenaturation than those containing a reducing sugar, such as lactose.

Formulations at pH 7 and those at pH 5.5 or 6 show an analogousdenaturation: it follows that, in the range considered, the influence ofpH value on the formulation stability seems negligible. In any case, pHvalues approaching or equalling neutrality are preferred for theformulations for injection.

The formulation selected for an in-depth study contains sucrose, at pH7. For the purpose of evaluating dosage influence on stability, twocompositions containing different quantities of active ingredients wereprepared (Table 7).

                  TABLE 7                                                         ______________________________________                                        Formulations of recombinant IL-6 with sucrose (content/vial)                  Comp.   Saccharose  Na.sub.2 HPO.sub.4 /NaH.sub.2 PO.sub.4                                                          IL-6                                    Form.   mg          mg       mg     pH  mg                                    ______________________________________                                        C1      48          0.313    0.336  7   0.035                                 C2      48          0.313    0.336  7   0.350                                 ______________________________________                                    

The investigation was carried out on samples stored in vials for 2, 4,8, and 10 weeks at 25° C., 37° C., and 50° C. Stability was measured byimmunologic assay expressed as per cent recovery of the sample activityat zero time (Table 8).

Table 9 recapitulates the stability of samples stored in vials for 4,10, and 12 weeks at the aforesaid temperatures. Stability was measuredby bioassay, still expressed as percent activity recovery at zero time.

                                      TABLE 8                                     __________________________________________________________________________    Study of the stability of IL-6 plus sucrose                                   formulations. Percent recovery vs. zero time - Immunologic assay              25° C.   37° C.                                                                             50° C.                                     Form.                                                                             2W 4W 8W 10W                                                                              2W 4W 8W 10W                                                                              2W 4W 8W 10W                                      __________________________________________________________________________    C1  105                                                                              90 94 97 111   94    101                                                                              95    90                                       C2     94 102      97 105      105                                                                              98                                          __________________________________________________________________________     W = weeks                                                                

                  TABLE 9                                                         ______________________________________                                        Study of the stability of IL-6 plus sucrose                                   formulations. Percent recovery vs. zero time - Bioassay                       25° C.  37° C.                                                                              50° C.                                     Form. 4W     10W    12W  4W   10W  12W  4W   10W  12W                         ______________________________________                                        C1    91     100    92   80   90        92   96   94                          C2    113                107            95                                    ______________________________________                                         W = weeks                                                                

As shown from the data of Tables 8 and 9, the denaturation of theformulations containing sucrose is extremely low and different IL-6dosages do not affect the formulation stability.

The very low denaturation of the aforesaid compositions was confirmed bychromatographic analyses conducted on samples at the same time intervalsand at the same temperatures as mentioned above. Chromatographicanalysis by molecular size separation was carried out with VARIANMICROPAK TSK GEL G-3000 SW column (diameter: 7.5 mm, length: 30 cm) at aflow rate of of 0.4 ml/min. The mobile phase was a 100 mM phosphatebuffer at pH 6.85 and 11.69 g/l NaCl.

The analyses did not show any variation of the samples chromatographicprofile in respect of zero time and confirmed that sucrose was the mostappropriate excipient for IL-6 formulations stabilization.

EXAMPLES OF PHARMACEUTICAL PRODUCTS

Materials: extra pure sucrose Ph fur, BP, Ph Nord, NF (Merck); reagentgrade Na₂ HPO₄.2H₂ O (Merck), NaH₂ PO₄.H₂ O (Merck); 0.1 M phosphoricacid (Merck); 0.1 M NaOH (Merck); water for injection. The containersused were DIN 2R glass vials (borosilicate glass, type I) sealed withPharmagummi butyl rubber and aluminium ring.

Preparation of IL-6 solution containing sucrose (for 1000 vialscontaining 35 μg IL-6/vial)

Saccharose (48 g), Na₂ HPO₄.2H₂ O (0.313 g) and NaH₂ PO₄.H₂ O (0.336 g)were dissolved in water For injection (400 ml) to form the initialsucrose solution. The obtained solution was divided into two equalparts. Recombinant IL-6 bulk (35 mg) was diluted with one solution partand adjusted to pH 7 with 0.1 M NaOH or H₃ PO₄. The two solutions werediluted to the final volume of 250 ml with water for injection.

The solution containing IL-6 was filtered through a 0.22μ Duraporesterile filter and diluted to the final volume with the remainingexcipients solution, filtered through the same Durapore filter. Duringthe process, the solution temperature was maintained at 4° C. to 8° C.

IL-6 solutions containing other excipients or a different quantity ofactive ingredient were prepared Following an analogous procedure.

Filling and freeze-drying

Vials were filled with 0.5 ml IL-6 solution, placed in the freeze-drier,and cooled to -45° C. for 3 to 6 hours. Freeze-drying started at -45° C.under 0.07 millibar vacuum. Heating scheme was as follows: +10° C. for10 to 12 hrs, then +30° C. until the cycle end.

The reconstituted solution was subjected to the usual quality controls.

Although the present invention has been illustrated by specificexamples, it is understood that variations to the applications describedherein can be introduced without falling outside the spirit and objectthereof.

We claim:
 1. A pharmaceutical composition containing an intimate solidmixture of Interleukin-6 (IL-6) and a stabilizing quantity of anonreducing sugar as stabilizing agent either alone or in conjuctionwith excipients, said intimate solid mixture being a freeze-driedpowder.
 2. The pharmaceutical composition according to claim 1 whereinsaid nonreducing sugar is sucrose or trehalose.
 3. The pharmaceuticalcomposition according to claim 1, wherein said IL-6 is recombinant. 4.The pharmaceutical composition according to claim 1, wherein saidstabilizing agent consists of sucrose.
 5. The pharmaceutical compositionaccording to claim 1, wherein said stabilizing agent is sucrose ortrehalose in combination with an amino acid.
 6. The pharmaceuticalcomposition according to claim 5 wherein said amino acid is arginine. 7.The pharmaceutical composition according to claim 1, wherein saidstabilizing agent is sucrose or trehalose in combination with albumin.8. The pharmaceutical composition according to claim 1, containing 35 or350 μg of IL-6 and 48 mg of sucrose.
 9. The pharmaceutical compositionaccording to claim 1, containing 35 or 350 μg of IL-6 and 47.5 mg oftrehalose.
 10. A hermetically closed sterile container containing thepharmaceutical composition according to claim
 1. 11. A solutioncontaining the solid mixture according to claim 1 in a solvent forinjection.
 12. In a process for the preparation of a solidpharmaceutical composition comprising the preparation of an aqueoussolution of the components for the composition, distribution of thesolution into containers and drying or freeze-drying of the solution inthe containers, the improvement wherein the components are a mixture ofInterleukin-6 and a stabilizing quantity of a non-reducing sugar. 13.The process according to claim 12, wherein the pH of the solution rangesbetween 5.5 and 7.0.
 14. The process according to claim 13 wherein thepH of the solution is
 7. 15. In a process for the preparation of a solidpharmaceutical composition comprising preparing an aqueous solution ofthe components for the pharmaceutical composition, drying orfreeze-drying the aqueous solution and distributing the solid mixtureobtained into containers, the improvement wherein the components are amixture of Interleukin-6 and a stabilizing quantity of a non-reducingsugar.
 16. The process according to claim 15, wherein the pH of thesolution ranges between 5.5 and 7.0.
 17. The process according to claim16, wherein the pH of the solution is 7.